ABSTRACT:
Inhibitors of reverse transcriptase (RT) of HIV-1 can be divided in two major groups, nucleoside analogue RT inhibitors such as zidovudine and lamivudine and non-nucleoside RT inhibitors (NNRTIs) such as nevirapine. Mechanistic aspects of inhibition have been studied extensively in cell-free assays using purified enzyme and defined nucleic acid substrates. Here, we have analyzed for the first time the effects of zidovudine and nevirapine during the initiation of plus-strand DNA synthesis, which requires accommodation of RNA-DNA as primer-template substrate. This particular reaction follows an ordered sequence of events: initiation, pausing after addition of the 12th DNA residue, RNAse H-mediated removal of the primer and continuation of DNA synthesis on DNA-DAN substrates. Since the entire process involves various structurally distinct complexes, RT inhibitors may act at each of these individual stages in a different manner.
In fact, we showed that nevirapine blocks the initiation of plus-strand DNA synthesis specifically at position +12. The NNRTI thus converts the natural pausing event into a termination site. We next showed that zidovudine-mediated chain termination is more efficient at early stages after initiation, when RT is still bound to the RNA-DNA substrate. In contrast, relatively high zidovudine/dTTP ratios are required to induce chain termination on homologous DNA-DNA substrates. Time course experiments revealed that chain-terminated products are efficiently synthesized, but disappeared with longer reaction time. In contrast, constant high levels of chain termination are observed with the corresponding RNA-DNA substrate. These data indicate that incorporation of stop nucleotides can occur with similar efficiencies on both types of substrates, but putative reverse reactions such as pyrophosphorolysis may rescue DNA synthesis more efficiently when the enzyme is complexed with DNA-DNA. To test this hypothesis directly, we compared the efficiency of pyrophosphorolysis on homologous DNA-DNA and RNA-DNA substrates and show that removal of terminal residues is drastically increased when DNA-DNA served as primer-template substrate.
